Detection of Gaseous Compounds by Needle Trap Sampling and

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Rapid Detection of Gaseous Compounds by Needle Trap Sampling and Direct Thermal-Desorption Photoionization Mass Spectrometry: Concept and Demonstrative Application to Breath Gas Analysis Juliane Kleeblatt, Jochen Klaus Schubert, and Ralf Zimmermann Anal. Chem., Just Accepted Manuscript • DOI: 10.1021/ac5039829 • Publication Date (Web): 17 Dec 2014 Downloaded from http://pubs.acs.org on December 20, 2014

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Analytical Chemistry

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Rapid Detection of Gaseous Compounds by Needle

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Trap Sampling and Direct Thermal-Desorption

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Photoionization Mass Spectrometry: Concept and

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Demonstrative Application to Breath Gas Analysis

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Juliane Kleeblatt,† Jochen Klaus Schubert ‡ and Ralf Zimmermann †*

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†

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University of Rostock, Dr.-Lorenz-Weg 1, 18059 Rostock, Germany and Comprehensive

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molecular analytics, Helmholtz Zentrum München, Ingolstädter Landstraße 1, 85764

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Neuherberg, Germany, ‡ Department of Anesthesia and Intensive Care, University of Rostock,

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Joint Mass Spectrometry Center, Chair of Analytical Chemistry, Institute of Chemistry,

Schillingallee 35, 18057 Rostock, Germany

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* Corresponding Author

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E-mail: [email protected], [email protected]. Phone:

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+49 (0) 381 498 6460. Fax: +49 (0) 381 498 118 6527.

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ACS Paragon Plus Environment

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KEYWORDS

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Needle-trap micro-extraction, gas chromatography, single photon ionization, resonance enhanced

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multiphoton ionization, time-of-flight mass spectrometry, alveolar sampling, propofol

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ABSTRACT

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A fast detection method to analyze gaseous organic compounds in complex gas mixtures was

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developed, using a needle trap device (NTD) in conjunction with thermal-desorption

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photoionization time-of-flight mass spectrometry (TD-PI-TOFMS). The mass spectrometer was

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coupled via a deactivated fused silica capillary to an injector of a gas chromatograph. In the hot

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injector the analytes collected on the NTD were thermally desorbed and directly transferred to

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the PI-TOFMS ion source. The molecules are softly ionized either by single photon ionization

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(SPI, 118 nm) or by resonance enhanced multiphoton ionization (REMPI, 266 nm) and the

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molecular ion signals are detected in the TOF mass analyzer. Analyte desorption and subsequent

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PI-TOFMS detection step only lasts ten seconds. The specific selectivity of REMPI (i.e. aromatic

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compounds) and universal ionization characteristics render PI-MS as a promising detection

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system. As a first, demonstrative application alveolar-phase breath gas of healthy, non-smoking

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subjects was sampled on NTDs. While smaller organic compounds as acetone, acetaldehyde,

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isoprene or cysteamine can be detected in the breath gas with SPI, REMPI depicts the aromatic

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substances phenol and indole at 266 nm. In breath gas of a healthy, smoking male subject,

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several xenobiotic substances such as benzene, toluene, styrene and ethylbenzene can be found

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as well. Furthermore, the NTD-REMPI-TOFMS setup was tested for breath gas taken from a


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mechanically ventilated pig under continuous intravenous propofol (2,6-diisopropylphenol,

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narcotic drug) infusion.

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INTRODUCTION

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Photoionization mass spectrometry (PI-MS) has been successfully used for on-line determination

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of substances in complex gaseous mixtures.1-3 However, if direct on-line sampling is not possible

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due to practical or legal constraints, analytes may be pre-concentrated onto adsorptive trapping

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devices, such as solid phase microextraction (SPME), and subsequently subjected to the analysis

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e.g. by GC/MS. Pawliszyn et al. developed the SPME method as an improved version of the

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solid phase extraction (SPE) technique. A fused silica fiber coated with polymeric adsorbent (e.g.

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polydimethylsiloxane or Carbowax/divinylbenzene) is exposed to the gaseous or liquid sampling

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medium and subsequently thermal desorbed solvent-free e.g. in a gas chromatographic injector.4,

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5

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technique, which allows easier handling during sampling and desorption. The NTD is a stainless

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steel needle packed with sorbent materials such as Carboxen, Carbopack, Tenax, divinylbenzene

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or polydimethylsiloxane (PDMS).6-11 Depending on the application the adsorbent material and

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arrangement (single- or multi-bed) can be adapted. The solvent-free method needs only small

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sampling volume; however, it is also possible to enhance the sensitivity by increasing the

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sampling volume. Therefore, the needle trap technique is restricted by breakthrough sampling

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volume and not by equilibration time or dynamics such as SPME. Thus, the sampling can be

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performed very fast. In this work the NTD sampling approach was combined with detection by

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photoionization mass spectrometry.

Further developments by Pawliszyn et al. had led to the more robust needle trap device (NTD)


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Photoionization comprises of single and multiphoton absorption/ionization processes (SPI and

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MPI). A neutral molecule is ionizable by single photon ionization (SPI) if the energy of a single

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vacuum-ultraviolet (VUV)-photon (λ < 200 nm) is equal to or higher than the ionization energy

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of the substance. Thus, if the photon energy is high enough the ionization technique is applicable

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to all organic substances. There are different possibilities to generate the required VUV-photons,

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which include deuterium discharge lamps, F2-lasers (157 nm), synchrotron radiation, electron

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beam pumped rare gas excimer lamps (EBEL) or frequency-tripled third harmonic generation of

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Nd:YAG lasers.12-15

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In contrast to the SPI process, MPI processes require much higher photon fluxes, achievable

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solely by pulsed lasers. For analytical purposes, resonance enhanced multiphoton processes

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(REMPI), where the photon energy is in resonance with an UV-spectroscopic state of the analyte

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molecules, is the most common method used. In case of a one-color, two-photon resonance

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enhanced multiphoton ionization process ((1+1)-REMPI, analytically most important REMPI

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process) a resonance absorption of a UV photon (i.e. gas phase UV spectroscopic step) is

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followed by the absorption of a second UV-photon of same laser pulse. If the sum of energies of

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both absorbed photons equals or exceeds the ionization threshold of the substance, the substance

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can be ionized with a rather high probability.16-18 REMPI and SPI mass spectrometry is known as

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a sensitive and selective analytical technique for fast on-line analysis of molecules in complex

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gaseous mixtures.

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Breath gas analysis stands out as an ideal, non-invasive diagnosis method. Although the breath

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gas is rather complex at the trace level, the matrix is comparably easy. Therefore, a complex,

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time and cost consuming sample preparation step is not necessary. Due to the direct correlation

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between substance concentration in blood and breath, analysis of breath gas may reduce the


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amount of invasive blood sampling steps. As the analysis of volatile organic compounds (VOCs)

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in breath is suspected to understand biochemical processes in the body, to allow the

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determination of disease types in clinical diagnosis, and to support the search for biomarkers, the

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research field is continuously increasing. Different on-line and off-line analytical methods have

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been developed.19-26 Due to the low concentrations of VOCs in breath, the already mentioned

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pre-concentration methods were utilized and commonly combined with gas chromatography

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mass spectrometry (GC-MS).27, 28 Due to the current trend to develop and apply soft ionization

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mass spectrometers for on-line breath gas analysis, several techniques are based on chemical

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ionization (CI).29 Examples include proton transfer reaction (time-of-flight) mass spectrometry

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(PTR-MS or PTR-TOFMS), selected ion flow tube mass spectrometry (SIFT-MS), ion mobility

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spectrometry (IMS) or extractive electrospray ionization mass spectrometry (EESI-MS).30-35

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However, rather new PI-MS methods have been rarely applied in the field of breath analysis until

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now. With SPI-TOFMS, compounds such as acetaldehyde, acetone, isoprene and phenol were

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on-line detected in breath gas of non-smokers. In smokers breath a large number of xenobiotic

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compounds are detectable. REMPI-TOFMS was applied for on-line measurements of aromatic

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substances in the mouth space of smokers and for detection of breath nitric oxide.36-38

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This publication reports on a new, fast coupling of needle trap devices (NTDs) and direct

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thermal-desorption photoionization time-of-flight mass spectrometry for the analysis of gas

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phase. The combination of trapping low concentrated volatile organic compounds, the sensitivity

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and selectivity of photoionization process as well as the fast measurement within ten seconds,

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qualifies the approach for rapid gas analysis. The experimental concept and setup is presented

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and the applicability to breath gas analysis is shown by first case study results. NTD-TD-PI-

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TOFMS was used for determination of substances in breath gas of healthy subjects (non-


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smoking and smoking) as well as in mechanically ventilated animal model. As an option, a gas

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chromatographic step was implemented for precise identification of measured compounds. The

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use of photoionization mass spectrometry as a detector method for GC is reported in literature as

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three-dimensional method (parameters: retention time, wavelength used for PI, molecular ion

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mass).39-44

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EXPERIMENTAL SECTION

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Needle Trap Devices. The used triple-bed NTDs were produced by Shinwa Ltd., Japan and were

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purchased from PAS Technology, Germany. The 22 gauge needles are 7 cm long with conical tip

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(figure 2) and filled with respectively 1 cm of polydimethylsiloxane (PDMS, 100/120 mesh),

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Carbopack B (60/80 mesh) and Carboxen 1000 (60/80 mesh). The very small grain sizes of the

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adsorbent materials were chosen to ensure the interaction of the analytes with the adsorbents and

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therefore, to prevent the channeling effect.45 Before using the NTDs, a conditioning step is

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essential to eliminate contaminations from production process. Therefore, the needles (20 pieces

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simultaneously) are flushed by a continuous helium flow and heated in a home-made solid

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aluminum block means ceramic heating plate (C-MAG HS7 IKAMAG, IKA) controlled by

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contact thermometer (ETS-D5). The conditioning temperature was 290 °C for 12 hours. Before

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every usage, the needles are conditioned for 30 min at 290 °C again. For storage and in-between

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usage each NTD is sealed with two Teflon caps.

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Instrumentation.

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a) Needle trap device thermal-desorption photoionization mass spectrometer (NTD-TD-PI-MS)


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The injector of an Agilent/HP 5890 gas chromatograph was used for thermal desorption of

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needle trap devices. The direct coupling to a photoionization time-of-flight mass spectrometer

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ensures a rapid measurement of the desorbed substances (figure 1). Within less than ten seconds,

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the sampled compounds are desorbed from the NTD at 290 °C in the injector, using SPME-GC

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inlet liner (Supelco, 78.5 mm × 6.5 mm × 0.75 mm) and being directly transferred in helium flow

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(Split: 1:2.5) via a deactivated heated fused silica capillary (BGB Analytik AG, 250 µm ID,

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350 µm OD, length 2.8 m, temperature 250 °C) to the ion source of the time-of-flight mass

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spectrometry. The used laser mass spectrometer (REMPI/SPI-TOFMS) was already described in

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detail.46, 47 Briefly, for REMPI the optical parametric oszillator (OPO) system (GWU-

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Lasertechnik GmbH, Erftstadt, Germany) is pumped by the third harmonic of a Nd:YAG

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laser (Continuum Surelite III, Santa Clara, USA, repetition rate 10 Hz) to generate UV laser

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pulses. The wavelength is tunable between 218 and 345 nm. For SPI, the third harmonic

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frequency (355 nm) of the same laser is used for direct third harmonic generation (THG) of

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118 nm pulses (photon energy: 10.49 eV) in a xenon filled gas cell. The laser beam with a

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wavelength of either 266 nm (REMPI) or 118 nm (SPI) was directly focused underneath the

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effusive molecular beam gas inlet. The generated ions were captured by electrostatic acceleration

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fields in the ion source of a reflectron time-of-flight mass spectrometer (Kaesdorf, Munich,

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Germany) and subsequently detected by a multichannel plate (MCP) detector. Data analysis was

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performed by home written LabView software tools.


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Figure 1. Measurement setup: needle trap thermal desorption in a hot gas chromatographic inlet

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coupled with photoionization (SPI/REMPI) time-of-flight mass spectrometer; direct and rapid

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measurement leads to simultaneous detection of substances in at most 10 seconds; variation with

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GC column provides detection after substance-specific retention time.

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b) Needle trap device thermal-desorption gas chromatography photoionization mass

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spectrometry (NTD-TD-GC-PI-MS)

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Optional it is possible to include a gas chromatographic step for identification of measured

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substances. In spite of losing the speed of the direct transfer method, the selectivity of REMPI

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and SPI-MS is enhanced by a further dimension. For this purpose, a DB-5MS capillary column

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(250 µm ID; 0.25 µm film thickness; J&W Scientific/Agilent Technologies, Santa Clara,

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California, USA) was used with different lengths and temperature programs for REMPI and SPI-

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TOFMS measurements. In case of REMPI, a 10 m column with a short temperature program

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(40 °C for 1 min, 12 °C/min to 250 °C for 1 min) was optimal. Due to the fact that also rather

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volatile compounds are detectable with SPI-TOFMS, the measurements were performed with a

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30 m long column and a modified temperature program (30 °C for 1 min, 10 °C/min to 250 °C


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for 1 min). An average of 5 mass spectra was used for GC measurements, thus, two data points

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per second were detected.

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Demonstration measurements: In Vivo Breath Gas Sampling. The healthy, smoking and non-

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smoking volunteering subjects were wearing a nose clip and breathe into a u-shaped device,

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which included a port for needle trap device and a connection to capnometer (Capnogard EtCO2

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Monitor, Novametrix Medical Systems INC.). Hence, the sampling was CO2-controlled, so that

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only the alveolar breath was pre-concentrated on NTD (see supporting information file available

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free of charge via the internet, figure S-1). The adsorption was managed by coupling a 1 ml

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single use syringe onto the Luer connection of the needle traps. Subsequently, the breath of the

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alveolar phase was pulled through the needle and pushed back again by agitating the syringe.

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The process was repeated for 30 times, hence, the sampling volume was 30 ml. Depending on

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the respiratory frequency of the subject, the sampling time was between 2 to 3 minutes. The

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average flow rate was approximately 25 to 30 ml/min.

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The volunteer smoker breathed into the device for two NTD samples, directly after smoking a

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cigarette. After one hour without smoking, again two breath samples were taken. All subjects did

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not eat nor brush their teeth for more than 1 hour before sampling.

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For the healthy volunteer group, two female (21 and 30 years old) and two male (23 and 29 years

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old) subjects were measured. The 49 years old male smoker indicated that he has been smoking

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for about 30 years and by now he smokes 30 to 40 cigarettes (1½-2 packs) per day.

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For determining propofol in breath gas, a pig of 25.6 kilos was mechanically ventilated

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undergoing surgery (supporting information figure S-2 b). The anesthesia was inducted with

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100 mg propofol (1%), 0.15 mg Fentanyl and 20 mg Nimbex. The intravenous infusion of


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propofol (2%) was maintained with a flow of 5 ml/h for about 8 hours. The alveolar sampling

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was realized connecting a t-piece into expiration tube and adsorbing respectively 20 ml of breath

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gas on four needle trap devices over a period of one hour. Additionally, an inspiration sample

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was taken from breathing hose.

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RESULTS AND DISCUSSION

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Evaluation of the developed NTD-TD-PI-MS coupling. After inserting the loaded needle trap

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device into the hot GC inlet, thermal desorption takes place and desorbed molecules are directly

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transferred into mass spectrometer within seconds. The desorption process itself lasts only a

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fraction of a second and thus a rather fast measurement is required in order to capture the

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dynamical behavior of the desorption process. The laser system operates with a repetition rate of

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10 Hz, thus, 10 mass spectra can be recorded per second. A sufficiently precise molecular signal

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vs. time curve of the desorption process could be recorded (supporting information figure S-

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2 a,b). The peak areas of these curves were used for quantification. The optimization before first

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measurements included the trial of split mode and according helium flow as well as selection of

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GC head pressure and temperatures of whole system. Best results were found for an injection

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split of 1:2.5, a head pressure of 50 kPa, an inlet temperature of 290 °C for PDMS/Carbopack

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B/Carboxen1000 packed needle trap device and a temperature of 250 °C for transfer capillary

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and ion source.

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The evaluation of the new coupled method was realized by using a gas standard mixture

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containing benzene (0.99 ppm), toluene (0.93 ppm) and 1,2,4-trimethylbenzene (1.3 ppm). The

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standard gas was diluted means evacuated 20 ml headspace vials so that every compound was in


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a concentration of approximately 100, 200, 300, 400 and 500 ppb. The differences in starting

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concentrations were recognized in acquisition (supporting information figure S-2 e,f). In each

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case 10 ml of the diluted standard were adsorbed on a needle trap device and desorbed in the GC

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inlet directly coupled to mass spectrometer. Each concentration was prepared and pre-

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concentrated four times and the limits of detection (LOD) and limits of quantitation (LOQ) were

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determined in accordance with DIN 32645 (table 1). Note that the signals and therefore, the

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limits of detection increase linear with rising sampling volume until the breakthrough volume of

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the respective substance is reached.

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Table 1. Limits of detection (LOD) and limits of quantitation (LOQ) of benzene, toluene and

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1,2,4-trimethylbenzene determined with REMPI and SPI using a sampling volume of 10 ml.

Compound

LOD REMPI

LOQ REMPI

LOD SPI

LOQ SPI

(ppb)

(µg)

(ppb)

(µg)

(ppb)

(µg)

(ppb)

(µg)

Benzene

14.1

49.1

60.4

210.5

30.3

105.6

124.0

432.1

Toluene

10.3

42.3

44.3

182.1

27.0

111.0

110.9

455.9

1,2,4-TMB

11.0

59.0

48.2

258.5

27.4

146.9

116.2

623.1

217 218

The results of standard gas mixture measurements show that the coupling is functioning

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reproducible and in a low concentration range. 10 ml of the diluted standard gas mixture are

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sufficient to obtain LOD in the lower ppb range for all substances and ionization methods.

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Performing a second desorption of the already desorbed needle trap showed that no carryovers

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were generated.

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More detailed information is given in supporting information file.


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Application Demonstration: Use of NTD-TD-PI-MS for fast breath gas analysis.

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a) Analysis of breath gas of healthy, non-smoking subjects

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The needle trap device thermal-desorption photoionization mass spectrometric method was

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applied to analyze the breath gas of healthy non-smoking subjects. According to their specific

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selectivity and sensitivity profile, the different photoionization methods REMPI and SPI address

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different breath gas analytes. Whereas SPI depicts more volatile substances such as acetone,

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acetaldehyde, isoprene and cysteamine, it is possible to detect the traces of aromatic breath gas

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compounds such as phenol and indole by REMPI. The named substances were confirmed by

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additional use of the gas chromatographic separation step (see below). Both techniques show

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background substances from needle trap adsorption material (figure 2). Thus, all mass spectra

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were corrected by blank mass spectra subtraction. Note that the spectra are corrected due to the

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variation of laser intensity for every measurement. Therefore, REMPI energy was measured

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behind ion source and the laser power was logged for SPI measurements.


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Figure 2. Mass spectra of breath gas of healthy, non-smoking subjects (2 female, 2 male) each

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measured by REMPI (266 nm) and SPI (118 nm). Left: REMPI measurements of respectively

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three averaged breath gas samples which are corrected by room air/needle trap background mass

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spectrum above; right: SPI measurements of respectively three averaged breath gas samples

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which are corrected by room air/needle trap background mass spectrum above.

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The averaged REMPI mass spectra of four healthy, non-smoking subjects (respectively three

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samples) are shown on the left in figure 2. Phenol and indole are detected in the breath gas of all

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subjects. Both substances have been described previously in literature as breath gas compounds,

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using time-intensive conventional enrichment and chemical analysis technologies. Indole

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(m/z 117) derives from catabolism of the amino acid tryptophan and decreases significantly in


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breath gas of patients with liver diseases because of higher levels of free tryptophan.48-50

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Additionally, it was found in connection with halitosis (bad breath), because of production from

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bacteria in mouth.51 The detected phenol (m/z 94) can arise endogenous from metabolism of

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aromatic amino acids. Statheropolous et al. described the determination of phenol in the breath

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gas of fasting subjects.52 Phenol, however, is contained in many foodstuffs (e.g. fruits, coffee)

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and thus, may also partly be of exogenous nature. The relation between signal intensities of both

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substances shows a variation depending on measured subject. All breath gas samples show in

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addition a yet unidentified molecular signal at m/z 212. Due to the fact that this signal is only

257

measurable by REMPI, the m/z 212 compound is of aromatic nature. Additionally, only for 21

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years old female a rather intense aromatic peak at m/z 148 was detected.

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The averaged SPI mass spectra are shown on the right side of figure 2. The detected acetone

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(m/z 58) is of endogenous origin since it was already described as decarboxylation product of

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acetoacetate, which is a result of dextrose metabolism and lipolysis, and acetyl-CoA.21 Acetone

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has been frequently detected in breath gas in a concentration range of more than 100 ppb up to a

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few thousand part-per-billion and often a relation between higher acetone concentration (up to

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ppm level) and diabetes was experienced.53-56 Furthermore, the endogenous substance

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acetaldehyde (m/z 44) is measurable in breath gas using single photon ionization. The compound

266

derives from ethanol metabolism in liver and was found in the ppb concentration range in breath

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gas of healthy persons.57 The endogenous origin of isoprene is not entirely clear, but it was

268

already described as a by-product of cholesterol biosynthesis and found in breath gas in a

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concentration range between a few ppb to a few hundred ppb.56, 58, 59 The compound with m/z 77

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was assigned to cysteamine. This assignation was confirmed by the GC analysis (NTD-TD-GC-

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SPI-MS, figure 4 a) with a standard. Cysteamine is the decarboxylation product of the amino


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acid cysteine and plays a decisive role in the cysteine metabolic cycle as product of synthesis and

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degradation of coenzyme A. Furthermore, the oxidation of cysteamine is indispensable for the

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production of hypotaurine and finally of taurine and that cysteamine is a major product of

275

metabolism of pantheinase.60, 61 Free, endogenous cysteamine was already found in tissues, e.g.

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of kidney and liver.62, 63 Due to the fact that cysteamine is difficult to detect in EI-MS (strong

277

fragmentation to m/z 30, after bond cleavage of C-C bond, NIST Chemistry WebBook) its

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occurrence in breath gas is not well covered in the literature. On-line mass spectrometry with soft

279

ionization techniques, such as the here used SPI, is preferred to detect cysteamine in breath gas.

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The assignation of m/z 77 to cysteamine, however, is supported by data from Martínez-Lozano et

281

al., who were able to detect the substance in breath gas using electrospray ionization and tandem

282

mass spectrometry.64

283

b) Analysis of breath gas of a healthy, smoking subject

284

The breath gas of the smoking person (figure 3) shows same substances as in breath from healthy

285

non-smokers (figure 2) and additional smoking related compounds. Most of the substances are of

286

exogenous origin, which can be clearly seen by comparing mass spectra obtained from sampling

287

directly after smoking with results obtained from samples one hour after smoking a cigarette. All

288

smoking related compounds result in lower signals or complete absence (below LOD) after one

289

hour. Same experiences were gained in literature mainly in terms of benzene, toluene, styrene,

290

ethylbenzene and xylenes.65-68 Poli et al. determined the breath of non-smokers, smokers and

291

subjects with lung diseases as lung cancer and described significantly higher concentrations of

292

ethylbenzene, xylenes, trimethylbenzene, toluene and benzene in breath gas of smoker compared

293

with non-smoker. Using 13 selected substances a discrimination of the groups (non-smoker,

294

smoker, NSCL, COPD) is possible.69 As marker for smoking 2,5-dimethylfuran (m/z 96) is


15


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Page 16 of 28

295

frequently described because of total absence in breath gas of healthy, non-smoking subjects.24,

296

68, 70, 71

297

substances not occurring in breath of non-smoker.

But also furan (m/z 68) and methyl furan (m/z 82) are known as smoking related

298 299

Figure 3. Mass spectra of breath gas of healthy, smoking subject (49 years, 20-40 cigarettes per

300

day) measured by REMPI (266 nm) and SPI (118 nm). Left: REMPI measurements of breath gas

301

samples (corrected by room air/needle trap background mass spectrum above) as well as

302

logarithmic scales for better presentation of small peaks; right: SPI measurements of breath gas

303

samples (corrected by room air/needle trap background mass spectrum above) as well as

304

logarithmic scales for better presentation of small peaks.


16

Page 17 of 28

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305

In figure 3 the differences between SPI (118 nm) and REMPI at a wavelength of 266 nm are

306

shown. While benzene (m/z 78) and toluene (m/z 92) are measurable by both techniques,

307

substances as styrene (m/z 104), ethylbenzene (m/z 106), 1,2,4-trimethylbenzene (m/z 120), m-,

308

p- and o-xylene (m/z 106) only were detected using REMPI. The NTD-TD-SPI-MS

309

measurement also shows signals at m/z 82 and m/z 96, which can be assigned to methylated and

310

dimethylated furan.36 The corresponding GC experiment (see below) indicates several isomers

311

for these masses. Directly after smoking, a great number of peaks result for both ionization

312

methods, but many exogenous compounds in the breath gas disappear during the hour without

313

smoking. With REMPI 266 nm (figure 3, left side) several peaks in the mass range from m/z 130

314

to m/z 200 appear directly after smoking. After only 1 hour almost all peaks had disappeared. A

315

similar pattern can be seen for SPI 118 nm (figure 3, right side). Especially, peaks with a mass-

316

to-charge ratio between 80 and 120 decrease in their intensity.

317

Confirmation of compound assignation: Including gas chromatographic separation. For

318

confirmation of the assignation of the detectable breath gas compounds, a gas chromatographic

319

step was implemented to use the three-dimensionality of the method (retention time, wavelength,

320

mass of molecular ion). Therefore, 10 µl of headspace above the appropriate pure substance were

321

put into an evacuated 20 ml headspace vial and afterwards the pressure was compensated by

322

helium. A sampling volume of 1 ml was adsorbed on needle trap device and subsequently

323

measured by presented NTD-GC-PI-TOFMS. The described procedure was repeated for every

324

substance with exception of acetaldehyde and acetone (1 µl headspace sufficient) as well as

325

cysteamine (10 ml headspace directly adsorbed on NTD). The several gas chromatograms of

326

relevant mass traces were merged to one complete chromatogram.


17


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327 328

Figure 4. NTD-TD-GC-TOFMS: Comparison of results for gas chromatographic measurements

329

of NTD smoker and non-smoker breath gas samples measured by a) SPI-TOFMS and b) REMPI-

330

TOFMS. Below: results for separately detected pure substances (combined chromatograms).

331

In figure 4 b) the comparison between the breath gas samples of non-smoker and smoker directly

332

and 1 hour after smoking measured with REMPI (266 nm) including gas chromatographic

333

separation is presented. Additionally, the determined gas chromatograms of the pure substances

334

are shown to demonstrate the successful identification. As discussed in last section the

335

exogenous smoking related compounds (benzene, toluene, ethylbenzene, styrene, xylenes)

336

decrease during 1 hour without smoking. Hence, the chromatograms nearly match the non-

337

smoking ones. Under the used GC conditions m- and p-xylene as well as o-xylene and styrene


18

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338

were not separated. The unknown substance at m/z 212 turns out to be at least five, potentially

339

isomeric compounds.

340

Showing the soft ionization, the mass spectra of detected compounds at the appropriate retention

341

time (room air, breath gas and pure substance) are presented in the supporting information (non-

342

smoker: S-3 to S-6 and smoker: S-7 to S-16).

343

In figure 4 a) the SPI chromatograms of breath gas of non-smoker and smoker (directly and 1

344

hour after smoking) as well as pure substances (below) are shown. As already seen in REMPI

345

measurements the smoking related compounds decrease; thus, the chromatograms assimilate to

346

non-smoking result. Despite the longer GC column and slower temperature program toluene and

347

cysteamine are eluting nearly at same retention time. Because of the strong tailing of acetone it is

348

not completely separated from acetaldehyde peak. The already mentioned m/z 77 appears at the

349

same retention time as the pure substance cysteamine. For m/z 82 three peaks result in

350

chromatogram, thus, probably those are the two possible methylfuran isomers and a further

351

unknown compound. Likewise, three peaks appear for m/z 96, which are possibly the isomers of

352

dimethylfuran (including 2,5-dimethylfuran) and ethylfuran. The substance at m/z 80 is also yet

353

unidentified.

354

A comparison between appearing substances in breath gas of non-smoker and smoker directly

355

after smoking is shown in table S-1 (supporting information) in terms of signal-to-noise ratios.

356

Substances occurring both in breath of non-smoking and smoking subject have mostly higher

357

signals in smoker’s odor. The table also shows the used photoionization method (SPI 118 nm or

358

REMPI 266 nm) at which the respective substance was detectable. In addition, the SPI mass


19


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Page 20 of 28

359

spectra at appropriate retention time are shown in supporting information file (non-smoker: S-17

360

to S-20 and smoker: S-21 to S-26).

361

Application of NTD-TD-PI-MS: Analysis of breath gas from mechanically ventilated

362

animal model. A further application of NTD-REMPI-TOFMS demonstrates that the narcotic

363

drug propofol can easily be detected in breath gas using a wavelength of 276.9 nm.72 The

364

detailed results are shown and discussed in the supporting information file.

365 366

CONCLUSIONS

367

In summary, a method and device for fast analysis of volatile compounds based on needle trap

368

device sampling and a direct in-injector thermal desorption photoionization time-of-flight mass

369

spectrometer was established and successful tested. A single measurement only takes some

370

seconds. With a special auto sampler device; thus, more than 100 measurements per hour would

371

be performable. Detection limits in the mid-lower ppb range were achieved with 10 ml gas

372

samples. For validation and compound identification purposes, a gas chromatographic step can

373

be optionally implemented.

374

The first demonstrative application on breath gas showed different substances detectable in the

375

single or multiphoton ionization mode. Using SPI, more volatile compounds as acetone,

376

acetaldehyde, isoprene and cysteamine are measurable in the current setup. REMPI (266 nm) is

377

especially suitable for aromatics; thus, it was possible to measure substances as phenol and

378

indole in breath gas. In case of healthy smoker, additionally exogenous substances such as

379

benzene, toluene, ethylbenzene, styrene, xylenes, 1,2,4-trimethylbenzene and benzaldehyde are


20

Page 21 of 28

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380

measurable. The breath gas measurements of medically ventilated pig undergoing surgery

381

demonstrate, that propofol is measurable in the breath gas of anaesthetized subjects using NTD-

382

TD-REMPI-TOFMS.

383

In comparison with vacuum ionization techniques (such as EI or PI, ionizing at 10-5 mbar) the

384

above mentioned CI-MS techniques such as PTR-MS or SIFT ionize at relatively high pressures

385

(10-3-10-1 mbar), which is beneficial for the achievable LOD: More ions can be produced in an

386

ionization process with given ionization agent concentrations and ionization cross sections in a

387

volume unit, if the pressure is higher and vice versa. The ionization pressure related LOD

388

advantage in CI-MS methods, however, is accompanied by an increased artifact-ion formation

389

(i.e. cluster- and adduct-ion formation or fragmentation). The use of the NTD sampling and

390

enrichment technology helps to compensate the inherent lower LOD of vacuum single photon

391

ionization (SPI). However, due to the reduced artifact formation and fragmentation as well as the

392

specific selectivity (REMPI), the application of PIMS has unique inherent advantages as well.

393

With SPI furthermore compounds, which are difficult detectable by CI based methods can be

394

addressed (e.g. alkanes).

395

Although we have not shown quantitative data for the breath gas demonstrative examples, the

396

method in principle can be used for quantitative analysis. In fact for real applications a sampling

397

system using a bleeding-in of an isotope labeled standard (e.g. 13C-toluene) would be used such

398

as for quantitation of on-line PIMS measurements.

399

In conclusion, the new coupled NTD-TD-SPI/REMPI-TOFMS method is promising and

400

suggests further development. The specific selectivity of the used ionization method allows

401

addressing volatile compounds which are not so easily detected by other on-line and off-line


21


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Page 22 of 28

402

methods. In the here chosen demonstrative application of breath gas analysis is, for example

403

cysteamine. Further research will concentrate on new application fields (e.g. combustion

404

effluents and process gases) and improving of limit of detection in the ppt concentration range by

405

using e.g. modern laser systems and optimized THG gas cells.

406 407

ACKNOWLEDGEMENT

408

The authors acknowledge Dr. Dennis Kleeblatt for synthesis and purification of propofol

409

metabolites as well as Janet Hofmann and Dr. Patricia Fuchs for assistance in sampling of breath

410

gas of animal model.

411

Funding Sources

412

This work was funded by the Interdisciplinary Faculty (INF) of the University of Rostock in

413

terms of a scholarship (J.K.).

414 415

SUPPORTING INFORMATION AVAILABLE

416

Additional information as noted in text. This material is available free of charge via the Internet

417

at http://pubs.acs.org.

418 419

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Detection of Gaseous Compounds by Needle Trap Sampling and

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